The invention relates to the overproduction of perlecan in transgenic animals and transfected animal cells as screening tools to identify lead therapeutics for the amyloid diseases.
The xe2x80x9camyloid diseasesxe2x80x9d consist of a group of clinically and generally unrelated human diseases which all demonstrate a marked accumulation in tissues of an insoluble extracellular substance known as xe2x80x9camyloidxe2x80x9d, and usually in an amount sufficient to impair normal organ function. Rokitansky in 1842 (Rokitansky, xe2x80x9cHandbuch der pathologischen Anatomiexe2x80x9d, Vol. 3, Braumuller and Seidel, Vienna) was the first to observe waxy and amorphous looking tissue deposits in a number of tissues from different patients. However, it wasn""t until 1854 when Virchow (Virchow, Arch. Path. Anat. 8:416 (1854)) termed these deposits as xe2x80x9camyloidxe2x80x9d meaning xe2x80x9cstarch-likexe2x80x9d since they gave a positive staining with the sulfuric acid-iodine reaction, which was used in the 1850""s for demonstrating cellulose. Although cellulose is not a constituent of amyloid, nonetheless, the staining that Virchow observed was probably due to the present of proteoglycans (PGs) which appear to be associated with all types of amyloid deposits. The name amyloid has remained despite the fact that Friederich and Kekule in 1859 discovered the protein nature of amyloid (Friedrich and Kekule, Arch. Path. Anat. Physiol. 16:50 (1859)). For many years, based on the fact that all amyloids have the same staining and structural properties, lead to the postulate that a single pathogenetic mechanism was involved in amyloid deposition, and that amyloid deposits were thought to be composed of a single set of constituents. Current research has clearly shown that amyloid is not a uniform deposit and that amyloids may consist of different proteins which are totally unrelated (Glenner, N. England J. Med. 302:1283-1292 (1980)).
Although the nature of the amyloid itself has been found to consist of completely different and unrelated proteins, all amyloids appear similar when viewed under the microscope due to amyloid""s underlying protein structure to adapt into a fibrillar structure. All amyloids regardless of the nature of the underlying protein 1) stain characteristically with the Congo red dye and display a classic red/green birefringence when viewed under polarized light (Puchtler et al, J. Histochem, Cytochem. 10:355-364 (1962)), 2) ultrastructurally consists of fibrils with a diameter of 7-10 nanometers and of indefinite length, 3) adopt a predominant beta-pleated sheet secondary structure. Thus, amyloid fibrils viewed under an electron microscope (30,000 times magnification) from the post-mortem brain of an Alzheimer""s disease patient would look nearly identical to the appearance of amyloid present in a biopsied kidney from a rheumatoid arthritic patient. Both these amyloids would demonstrate a similar fibril diameter of 7-10 nanometers.
In the mid to late 1970""s amyloid was clinically classified into 4 groups, primary amyloid, secondary amyloid, familial amyloid and isolated amyloid. Primary amyloid, is amyloid appearing de novo, without any preceding disorder. In 25-40% of these cases, primary amyloid was the antecedent of plasma cell dysfunction such as the development of multiple myeloma or other B-cell type malignancies. Here the amyloid appears before rather than after the overt malignancy. Secondary amyloid, appeared as a complication of a previously existing disorder. 10-15% of patients with multiple myeloma eventually develop amyloid (Hanada et al., J. Histochem. Cytochem. 19:1-15 (1971)). Patients with rheumatoid arthritis, osteoarthritis, ankylosing spondylitis can develop secondary amyloidosis as with patients with tuberculosis, lung abscesses and osteomyelitis (Benson and Cohen, Arth. Rheum. 22:36-42 (1979); Kamei et al., Acta Path. Jpn. 32:123-133 (1982); McAdam et al., Lancet 2:572-575 (1975)). Intravenous drug users who self-administer and who then develop chronic skin abscesses can also develop secondary amyloid (Novick, Mt. Sin. J. Med. 46:163-167 (1979)). Secondary amyloid is also seen in patients with specific malignancies such as Hodgkin""s disease and renal cell carcinoma (Husby et al., Cancer Res. 42:1600-1603 (1982)). Although these were all initially classified as secondary amyloid, once the amyloid proteins were isolated and sequenced many of these turned out to contain different amyloid proteins.
The familial forms of amyloid also showed no uniformity in terms of the peptide responsible for the amyloid fibril deposited. Several geographic populations have now been identified with genetically inherited forms of amyloid. One group is found in Israel and this disorder is called Familial Mediterranean Fever and is characterized by amyloid deposition, along with recurrent inflammation and high fever (Mataxas, Kidney 20:676-685 (1981)). Another form of inherited amyloid is Familial Amyloidotic Polyneuropathy, and has been found in Swedish (Skinner and Cohen, Biochem. Biophys. Res. Comm. 99:1326-1332 (1981)), Portuguese (Saraiva et al., J. Lab. Clin. Med. 102:590-603 (1983); J. Clin. Invest. 74:104-119 (1984)) and Japanese (Tawara et al., J. Lab. Clin. Med. 98:811-822 (1981)) nationalities. Amyloid deposition in this disease occurs predominantly in the peripheral and autonomic nerves. Hereditary amyloid angiopathy of Icelandic origin is an autosomal dominant form of amyloid deposition primarily affecting the vessels in the brain, and has been identified in a group of families found in Western Iceland (Jennson et al., Clin. Genet. 36:368-377 (1989)). These patients clinically have massive cerebral hemorrhages in early life which usually causes death before the age of 40.
The primary, secondary and familial forms of amyloid described above tend to involve many organs of the body including heart, kidney, liver, spleen, gastrointestinal tract, skin, pancreas, and adrenal glands. These amyloid diseases are also referred to as xe2x80x9csystemic amyloidsxe2x80x9d since so many organs within the body demonstrate amyloid accumulation. For most of these amyloidoses, there is no apparent cure or effective treatment and the consequences of amyloid deposition can be detrimental to the patient. For example, amyloid deposition in kidney may lead to renal failure, whereas amyloid deposition in heart may lead to heart failure. For these patients, amyloid accumulation in systemic organs leads to eventual death generally within 3 to 5 years.
Isolated forms of amyloid, on the other hand, tend to involve a single organ system. Isolated amyloid deposits have been found in the lung, and heart (Wright et al., Lab. Invest. 30:767-773 (1974); Pitkanen et al., Am. J. Path. 117:391-399 (1984)). Up to 90% of type II diabetic patients (non-insulin dependent form of diabetes) have isolated amyloid deposits in the pancreas restricted to the beta cells in the islets of Langerhans (Johnson et al., New Engl. J. Med. 321:513-518 (1989); Lab Invest 66:522-535 (1992)). Isolated forms of amyloid have also been found in endocrine tumors which secrete polypeptide hormones such as in medullary carcinoma of the thyroid (Butler and Khan, Arch. Path. Lab. Med. 110:647-649 (1986); Berger et al., Virch. Arch. A Path. Anat. Hist. 412:543-551 (1988)). A serious complication of long term hemodialysis is amyloid deposited in the medial nerve and clinically associated with carpal tunnel syndrome (Gejyo et al., Biochem. Biophys. Res. Comm. 129:701-706 (1985); Kidney Int. 30:385-390 (1986)). By far, the most common type and clinically relevant type of organ-specific amyloid, and amyloid in general, is that found in the brains of patients with Alzheimer""s disease (see U.S. Pat. No. 4,666,829 and Glenner and Wong, Biochem. Biophys. Res. Comm. 120:885-890 (1984); Masters et al., Proc. Natl. Acad. Sci. USA 82:4245-4249 (1985)). In this disorder, amyloid is predominantly restricted to the central nervous system. Similar deposition of amyloid in the brain occurs in Down""s syndrome patients once they reach the age of 35 years (Rumble et al., New England J. Med. 320:1446-1452 (1989); Mann et al., Neurobiol. Aging 10:397-399 (1989)). Other types of central nervous system amyloid deposition include rare but highly infectious disorders known as the prion diseases which include Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, and kuru (Gajdusek et al., Science 197:943-960 (1977); Prusiner et al., Cell 38:127-134 (1984); Prusiner, Scientific American 251:50-59 (1984); Prusiner et al., Micr. Sc. 2:33-39 (1985); Tateishi et al., Ann. Neurol. 24:35-40 (1988)).
It was misleading to group the various amyloidotic disorders strictly on the basis of their clinical features, since when the major proteins involved were isolated and sequenced, they turned out to be different. For example, amyloid seen in rheumatoid arthritis and osteoarthritis, now known as AA amyloid, was the same amyloid protein identified in patients with the familial form of amyloid known as Familial Mediterranean Fever. Not to confuse the issue, it was decided that the best classification of amyloid should be according to the major protein found, once it was isolated, sequenced and identified.
Thus, amyloid today is classified according to the specific amyloid protein deposited. The amyloid diseases include, but are not limited to, the amyloid associated with Alzheimer""s disease, Down""s syndrome and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (wherein the specific amyloid is now known as the beta-amyloid protein or Axcex2), the amyloid associated with chronic inflammation, various forms of malignancy and Familial Mediterranean Fever (AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell abnormalities (AL amyloid), the amyloid associated with type II diabetes (amylin or islet amyloid), the amyloid associated with the prion diseases including Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru and animal scrapie (PrP amyloid), the amyloid associated with long-term hemodialysis and carpal tunnel syndrome (beta2-microglobulin amyloid), the amyloid associated with senile cardiac amyloid and Familial Amyloidotic Polyneuropathy (prealbumin or transthyretin amyloid), and the amyloid associated with endocrine tumors such as medullary carcinoma of the thyroid (variants of procalcitonin).
Although there are many different types of amyloid diseases as described above, there are only a few animal models for these diseases to develop new therapeutic agents. Inflammation-associated or AA amyloidosis has a well-defined experimental mouse model which is used to induce amyloid deposition in systemic organs such as spleen, liver and kidney (Snow and Kisilevsky, Lab. Invest. 53:37-44 (1985)). In addition, only some of the Alzheimer""s disease neuropathology is observed in current animal models of the disease (described below). Thus, there is a need for the development of new transgenic animal models for the amyloid diseases including Alzheimer""s disease. In addition, there is a need for new cell culture models to rapidly screen and identify new lead therapeutics for each of the amyloid diseases.
The invention provides a transgenic non-human animal expressing a perlecan encoding transgene. Also provided is a double-transgenic non-human animal expressing a perlecan and a amyloid encoding transgene. A method of screening for a compound which alters the rate or extent of amyloid deposition is additionally provided. The method consists of: (a) constructing a perlecan transgenic animal; (b) administering an effective amount of a test compound to said perlecan transgenic animal; and (c) determining whether said test compound alters the extent or rate of amyloid deposition. Finally, the invention provides a method of screening for a compound which alters the rate or extent of amyloid deposition. The method consists of: (a) constructing a perlecan/amyloid double-transgenic animal; (b) administering an effective amount of a test compound to said perlecan/amyloid double-transgenic animal; and (c) determining whether said test compound alters the extent or rate of amyloid deposition.